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Impact of Urea on Monoclonal Antibodies: Multiple Destabilization and Aggregation Effects for Therapeutic Immunoglobulin G Proteins.

ABSTRACT: We performed nano differential scanning fluorimetry (nanoDSF) measurements of immunoglobulin G (IgG) in urea gradient solutions under thermal unfolding. Our results show that the denaturing effect of urea on individual IgG domains can be monitored via a linear mapping of thermal shift curves to the corresponding urea concentrations. Assignment of IgG domains to each thermal shift curve allows for a reliable differentiation of the underlying mechanisms. Further results show a decisive influence of salt-induced electrostatic screening effects. We are able to explain all findings by preferential binding mechanisms in combination with electrostatic effects. The results of our study shed more light on the complex interaction mechanisms between buffer solutions and complex proteins, which are important for improving the shelf life of protein therapeutic formulation.

SUBMITTER: Yang JY

PROVIDER: S-EPMC10851413 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Impact of Urea on Monoclonal Antibodies: Multiple Destabilization and Aggregation Effects for Therapeutic Immunoglobulin G Proteins.

Yang Ji Young JY Burkert Oliver O Mizaikoff Boris B Smiatek Jens J

ACS omega 20240126 5

We performed nano differential scanning fluorimetry (nanoDSF) measurements of immunoglobulin G (IgG) in urea gradient solutions under thermal unfolding. Our results show that the denaturing effect of urea on individual IgG domains can be monitored via a linear mapping of thermal shift curves to the corresponding urea concentrations. Assignment of IgG domains to each thermal shift curve allows for a reliable differentiation of the underlying mechanisms. Further results show a decisive influence o ...[more]

PMID: 38343970

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Project description:The monospecific and bivalent characteristics of naturally occurring immunoglobulin G (IgG) antibodies depend on homodimerization of the fragment crystallizable (Fc) regions of two identical heavy chains (HCs) and the subsequent assembly of two identical light chains (LCs) via disulfide linkages between each HC and LC. Immunoglobulin Fc heterodimers have been engineered through modifications to the CH3 domain interface, with different mutations on each domain such that the engineered Fc fragments, carrying the CH3 variant pair, preferentially form heterodimers rather than homodimers. Many research groups have adopted different strategies to generate Fc heterodimers, with the goal of high heterodimerization yield, while retaining biophysical and biological properties of the wild-type Fc. Based on their ability to enforce heterodimerization between the two different HCs, the established Fc heterodimers have been extensively exploited as a scaffold to generate bispecific antibodies (bsAbs) in full-length IgG and IgG-like formats. These have many of the favorable properties of natural IgG antibodies, such as high stability, long serum half-life, low immunogenicity, and immune effector functions. As of July 2016, more than seven heterodimeric Fc-based IgG-format bsAbs are being evaluated in clinical trials. In addition to bsAbs, heterodimeric Fc technology is very promising for the generation of Fc-fused proteins and peptides, as well as cytokines (immunocytokines), which can present the fusion partners in the natural monomeric or heterodimeric form rather than the artificial homodimeric form with wild-type Fc. Here, we present relevant concepts and strategies for the generation of heterodimeric Fc proteins, and their application in the development of bsAbs in diverse formats for optimal biological activity. In addition, we describe wild-type Fc-fused monomeric and heterodimeric proteins, along with the difficulties associated with their preparations, and discuss the use of heterodimeric Fc as an alternative scaffold of wild-type Fc for naturally monomeric or heterodimeric proteins, to create Fc-fusion proteins with novel therapeutic modality.

Dataset Information

Impact of Urea on Monoclonal Antibodies: Multiple Destabilization and Aggregation Effects for Therapeutic Immunoglobulin G Proteins.

Publications

Impact of Urea on Monoclonal Antibodies: Multiple Destabilization and Aggregation Effects for Therapeutic Immunoglobulin G Proteins.

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